The present invention relates to the field of biotechnology, and more particularly to that of industrial fermentations by recombinant microorganisms.
Still more particularly, it relates to a host/vector pair which is highly stable in a complex medium, its preparation and its use in industrial fermentation.
The advances accomplished in the field of molecular biology have made it possible to modify microorganisms in order to make them produce specific recombinant proteins, preferably heterologous proteins. In particular, numerous genetic studies have been performed on the bacterium E. coli. More recently, yeasts such as Saccharomyces, Kluyveromyces, Pichia, or even Hansenula, have emerged as promising host organisms for this mode of protein production.
However, the industrial application of these new modes of production is still limited, especially by the problems of the efficacy of gene expression in these recombinant microorganisms, and by the difficulty of obtaining recombinant cells which are stable under industrial fermentation conditions. One of the essential operational constraints is indeed linked to the segregational stability of an expression vector inside the host used. At the industrial level, a vector should possess a high stability over at least 25 successive generations, which approximately represent the number of generations required to go as far as the end of a 200-m3 fed batch-type industrial fermenter (Principles of Fermentation Technology, Stanburry and Whitaker, Pergamon Press, Oxford, 1984). The stability of the vector must be even higher in the case of continuous fermentation where it must reach not less than about one hundred generations.
In bacteria, the most common solution used in the laboratory consists of inserting a gene for resistance to an antibiotic into the plasmid used, which endows the bacteria with the capacity to survive and grow in a selective medium containing said antibiotic. However, because of security and regulatory constraints in the field of biotechnology, it is essential to be able to avoid the use of antibiotic resistance genes at the industrial level. In yeasts, the most commonly used method consists of culturing cells with a defective pathway for the biosynthesis of amino acids (Trp, Leu, His) or of purine (adenine) or pyrimidine (uracil) bases, said cells being transformed by a vector containing a gene which is capable of complementing this defect. However, this approach requires the use of media lacking the amino acid or the base for which the host strain is auxotrophic. The use of such synthetic media has numerous disadvantages. In particular, these media are expensive, which is incompatible with an industrial use, and furthermore, they lead to slower growth of the cells and to a smaller biomass.
A solution has been proposed to avoid the use of a synthetic medium or of antibiotic resistance genes, which consists of (i) mutating a gene which is essential for survival in a complex medium in the host cell and (ii) introducing an intact copy of said gene into the expansion plasmid used. This system, the principle of which is to force the host cell to retain its plasmid, has enabled the stability of the host/vector pair to be increased. This system has, in particular, been described for E. coli, for the dapD gene which encodes tetrahydropicolinate-N-succinyl transferase (EP 258 118), for the va1S gene whose product is an enzyme which is required for protein synthesis (Skogman and Nilsson, Gene 31 (1984) 117), and for the ssb gene whose product is essential for DNA replication and for the survival of the cell (Porter et al., Bio/technology vol. 8 (1990) 47). Ferrari et al. have also described the use of the racemase alanine gene for stabilising a plasmid inside a B. subtilis mutant in which this gene was not functional (Bio/technology vol. 3 (1985) 1003). Application WO 86/01224 describes a similar selection system which is suitable for the yeast S. cerevisiae. This system uses the yeast S. cerevisiae which has a mutation in 2 genes which are involved in the biosynthesis of uracil. It consists of (i) inactivating one of the genes for the synthesis of uracil, (ii) transforming said cell with a vector carrying the active gene, and (iii) blocking the other metabolic pathway by mutagenesis.
Another approach for obtaining expression systems which are stable in complex media consists of using vectors which are integrated into the genome of the host cell. However, this system enables only a small number of copies of the vector to be obtained per recombinant cell, and furthermore, the transformation frequency is low. Under these conditions, the levels of expression of heterologous genes are not always satisfactory. A method enabling amplification of a gene which is integrated into the genome has, moreover, been developed in S. cerevisiae, by directing integration towards the genes encoding ribosomal proteins, said genes being present in multiple copies in the genome. However, this system proves to be unstable when the integrated genes are expressed at high levels, whether they are homologous or heterologous genes.
Currently, the use of the new yeasts, different from S. cerevisiae, for the production of recombinant proteins requires the development of tools which are adapted to these microorganisms, in order to resolve in particular the problems of stability of expression vectors for heterologous genes. More specifically, yeasts which are taxonomically related to the Kluyveromyces genus appear to possess a particularly advantageous capacity for secreting recombinant proteins. This has been observed in particular in the case of the yeast K. lactis, for the production of chymosin (EP 96430), IL-1xcex2 or human serum albumin (EP 361991). However, no sufficiently stable multicopy expression vectors exist in this organism to permit its use in industrial processes. In particular, no vectors exist which are stable in complex media, enabling large-scale processes, especially continuous processes, to be envisaged using this organism. Indeed, although certain vectors which are stable in K. lactis have been described (EP 361991), the introduction of a heterologous gene expression cassette into these vectors produces a substantial destabilising effect, especially under conditions for inducing production.
A particularly efficient means for stabilising host/vector pairs in which the host is a yeast of the Kluyveromyces genus, has now been found.
One embodiment of the invention consists of a host/vector pair which is highly stable in a complex medium, characterised in that the host is a yeast of the Kluyveromyces genus in which a gene which is essential for its growth in said medium is nonfunctional, and in that the vector carries a functional copy of said gene.
Within the context of the present invention, complex medium is understood to mean any medium for industrial fermentation which is compatible with the economic constraints of a large-scale operation. In particular, it relates to media containing industrial-type raw materials: maize soluble extract, yeast extract, molasses or xe2x80x9cdistillersxe2x80x9d, for example, as opposed to defined synthetic media which are supplemented (for example with antibiotics). However, it is understood that the present invention may also be used on synthetic media, although this embodiment is less advantageous.
Moreover, it is understood that the functional gene which is present in the vector may be a homologous or heterologous gene.
Genes which are essential for the growth of the host cell in a complex medium include genes which are involved in the metabolism of a carbon source present in the medium (galactose, lactose, glucose and the like), and genes participating in cellular division, in membrane synthesis, in protein synthesis or DNA replication or transcription.
More preferably, the invention consists of a host/vector pair which is highly stable in a complex medium, characterised in that the host is a yeast of the Kluyveromyces genus in which a gene which is involved in glycolysis is nonfunctional, and in that the vector carries a functional copy of said gene.
It has indeed been shown that host/vector pairs which are very stable in a complex medium, which is compatible with an industrial operation, may be obtained in Kluyveromyces by rendering the host cell dependent on its plasmid when it has to call into play a glycolysis step in order to metabolise the carbon sources of the medium.
More preferably, the present invention relates to host/vector pairs in which the host is chosen from the yeasts Kluyveromyces lactis and Kluyveromyces fragilis.
In yeasts, glycolysis involves a succession of complex enzymatic and chemical steps leading to the formation of molecules of ATP and ethanol. The main enzymes involved in this pathway are known and some of the genes encoding these enzymes have been identified and cloned: the genes encoding phosphofructokinase (Kopperschlager et al., Eur. J. Biochem. 81 (1977) 317); pyruvate kinase (Aust et al., J. Biol. Chem. 253 (1978) 7508); phosphoglycerate kinase (Scopes, Meth. Enzymol. 42 (1975) 134); phosphoglycerate mutase (Price et al., FEBS Letters 143 (1982) 283); triose phosphate isomerase (Alber et al., J. Biol. Chem. 256 (1981) 1356); pyruvate decarboxylase (Gounarb et al., Biochim. Biophys. Acta 405 (1975) 492), and phosphoglucose isomerase (Noltmann, The enzymes, vol VI, Academic Press, N.Y., 271, 1972) have been identified in S. cerevisiae. Some glycolytic genes have been cloned into K. lactis. They are, more specifically, the RAG1 and RAG2 genes which encode a sugar-transporting protein (Goffrini et al., Nucl. Acid. Res. 18 (1990) 5294) and a phosphoglucose isomerase (Wxc3xa9solowski-Louvel, Nucl. Acid. Res. 16 (1988) 8714), respectively; and genes which encode alcohol dehydrogenases, ADH (Saliola et al., Yeast 6 (1990) 193-204; Saliola et al., Yeast 7 (1991) 391-400).
However, these genes cannot be efficiently used for stabilising expression vectors in yeasts of the Kluyveromyces genus. Indeed, the RAG1 gene and ADH genes are not essential for the use of glucose. Furthermore, earlier work on Kluyveromyces has shown that the RAG2 gene, whose PGI equivalent is known to be essential for the growth of the yeast S. cerevisiae on a glucose-containing medium, was not essential in Kluyveromyces (Wxc3xa9solowski-Louvel, as mentioned above; Goffrini et al., Yeast 5 (1989) 99-106). This result indicated a difference in behaviour for the Kluyveromyces and Saccharomyces yeasts with respect to the use of glucose, which did not enable the production, in Kluyveromyces, of a stable host/vector pair by using glycolytic genes as selection markers.
Under these conditions, the use of these genes for the stabilisation of expression vectors in yeasts of the Kluyveromyces genus was neither described nor suggested, nor possible.
Surprisingly, the applicant has now shown that some glycolytic genes may be essential for the growth and/or survival of yeasts of the Kluyveromyces genus in a complex medium and that they may enable the production of particularly stable host/vector pairs. In particular, stable and efficient systems may be obtained when the glycolytic gene chosen is a single gene whose product is essential for the metabolism of the carbon sources of the medium by this yeast. Indeed, some glycolysis steps involve activities which may be encoded by several genes. This is the case, especially in S. cerevisiae, for enolase (ENO), phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or alcohol dehydrogenase (ADH) activities which are encoded by several genes. In this case, inactivation of one of these enzymatic activities would require the inactivation of all the genes which are capable of encoding it.
Preferably, in the host/vector pair of the invention, the gene involved in glycolysis is a single gene whose product is essential for the metabolism of the carbon sources of the medium by the yeast.
Still more preferably, it is a gene which is chosen from the genes encoding phosphoglycerate kinase (PGK), phosoglycerate mutase (GPM), pyruvate kinase (PYK) and triose phosphate isomerase (TPI).
The selected gene may be rendered nonfunctional in the host yeast in various ways. It is possible to use nonspecific mutagenesis techniques. The yeasts may be treated with physical agents (X rays; ultra violet rays and the like) or chemical agents (intercalating agents, mono- or bialkylating agents and the like). The yeasts thus treated are then selected on various media depending on the desired mutation.
It is also possible to use specific mutagenesis tools, especially techniques for mutational insertions into DNA or for gene replacement by homologous recombination (Rothstein, Meth. Enzymol. 101 (1983) 202). To this effect, at least 2 mutagenesis pathways are possible:
replacing the gene to be deleted by a dominant selection marker of the antibiotic resistance marker type (geneticin, fluomycin and the like). It has been possible to apply this strategy directly to a wild strain;
replacing the gene to be deleted by an intact copy of a gene complementing an auxotrophy (ura3, trp1, leu2, and the like) of the strain used. This strategy may be applied to any strain exhibiting an auxotrophy, or to a wild strain previously made auxotrophic.
Preferably, in the host/vector pair of the invention, the functional copy of the essential gene present in the vector is placed under the control of a weal promoter. This advantageous embodiment enhances the stability of the pair and the increase in the number of copies of the vector per host cell and, consequently, tends to increase the level of expression of a recombinant gene. Examples of weak promoters which may be used for this purpose include the bidirectional promoter of the killer toxin gene (Tanguy-Rougeau et al., Gene 91 (1990) 43) or that of a heterologous gene such as the promoter of the acid phosphatase gene of S. cerevisiae under repression conditions (phosphate-containing culture medium). In another preferred embodiment of the invention, the functional copy of the essential gene which is present in the vector is either completely free of promoter or is placed under the control of a defective promoter, whether as a result of a mutation of the promoter itself or as a result of the inactivation of a gene involved in the transcriptional activation of said promoter. In another embodiment of the invention, the essential gene present in the vector may be a gene which is defective under certain conditions such as temperature conditions, for example. In particular, it may be a heat-sensitive gene.
Preferably, in the host/vector pair of the invention, the vector comprises, in addition, a DNA sequence containing a structural gene encoding at least a desired protein, and signals permitting its expression.
In a preferred embodiment of the invention, the structural gene encodes a protein which is important in the pharmaceutical or agri-foodstuffs industries.
Structural genes include, but are not limited to, enzymes (such as, in particular, superoxide dismutase, catalase, amylases, lipases, amidases, chymosin and the like), blood derivatives (such as serum albumin or variants or precursors thereof, alpha- or beta-globin, factor VIII, factor IX, the von Willebrand factor or portions thereof, fibronectin, alpha-1-antitrypsin and the like), insulin and its variants, lymphokines [such as interleukins, interferons, colony stimulating factors (G-CSF, GM-CSF, M-CSF and the like), TNF, TRF, MIP and the like], growth factors (such as growth hormone, erythropoietin, FGF, EGF, PDGF, TGF and the like), apolipoproteins, antigenic polypeptides for the production of vaccines (hepatitis, cytomegalovirus, Epstein-Barr, herpes and the like), viral receptors, or even fusions of polypeptides such as in particular fusions containing an active portion fused with a stabilising portion (for example, fusions between albumin or fragments of albumin and the receptor or a portion of a virus receptor (CD4 and the like)).
Advantageously, moreover, the DNA sequence comprises, in addition, signals enabling the secretion of the recombinant protein. These signals may correspond to the natural signals for the secretion of the protein in question, but they may also be of a heterologous origin. In particular, secretion signals derived from yeast genes such as those from the killer toxin or alpha pheromone gene may be used.
Preferably, the structural gene encodes human serum albumin, its precursors or its molecular variants. xe2x80x9cMolecular variantsxe2x80x9d of albumin is understood to mean the natural variants resulting from the polymorphism of albumin, the structural derivatives possessing an albumin-type activity, the truncated forms of albumin, or any albumin-based hybrid protein.
In another embodiment, the structural gene(s) encodes(s) polypeptides which are involved at the genetic or biochemical level in the biosynthesis of a metabolite. In particular, they may be genes which are involved in the biosynthesis of amino acids, antibiotics or vitamins.
Generally, the signals enabling the expression of the structural gene are chosen from transcription promoters and terminators. It is understood that these signals are chosen as a function of the structural gene and of the desired result. In particular, it may be preferable to use in certain cases a promoter which can be regulated so as to be able to uncouple the host growth phase(s) from the gene expression phase. Likewise, for reasons related to strength and compatibility, it may be preferable to use, in certain cases, the natural promoters for the structural genes and, in other cases, promoters of a different origin.
Preferably, the promoters used are derived from yeast genes and, still more preferably, from yeast glycolytic genes. The promoters derived from the glycolytic genes of yeasts of the Saccharomyces or Kluyveromyces genus are of very particular importance. In particular, examples include the promoters of genes which encode phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), enolases (ENO) or alcohol dehydrogenases (ADH). Promoters derived from genes which are strongly expressed, such as the lactase gene (LAC4), acid phosphatase gene (PHO5) or gene for elongation factors (TEF) may also be mentioned.
Moreover, these promoter regions may be modified by mutagenesis, for example, so as to add additional elements for the control of transcription, such as in particular UAS regions (xe2x80x9cUpstream Activating Sequencexe2x80x9d). By way of example, a hybrid promoter between the promoters of the PGK and GAL1/GAL10 genes of S. cerevisiae gives good results.
The host/vector pairs of the invention may be used in methods for producing recombinant proteins. They thus enable particularly efficient production systems to be achieved in yeasts of the Kluyveromyces genus.
In this respect, another embodiment of the invention relates to a method for producing a recombinant protein in which a host/vector pair as defined above, comprising the structural gene which encodes said protein under the control of signals permitting its expression, is cultured and the protein produced is recovered.
Such a method enables the production of proteins which are important in the pharmaceutical or agri-foodstuffs industries, such as those stated above. It is particularly suitable for the production of human serum albumin, its precursors and molecular variants, although not limited thereto.
The host/vector pairs of the invention may also be used directly as catalysts in bioconversion reactions.
Another subject of the invention relates to the expression vectors for yeasts of the Kluyveromyces genus carrying a functional copy of a gene which is essential for the growth of the Kluyveromyces yeast on a complex medium.
More preferably, the essential gene is a gene which is involved in one of the above-mentioned functions. This gene may be obtained by any method known to a person skilled in the art (hybridisation cloning using heterologous probes, mutant complementation cloning, and the like).
Advantageously, the vectors of the invention are free of any bacterial sequences. It has indeed been shown that it is possible to transform Kluyveromyces yeasts with such vectors in vitro. This system has the advantage of enabling the use of vectors which are smaller and therefore easier to manipulate and capable of accepting larger recombinant DNA sequences.
Examples of such vectors include, in particular, the vectors pYG1023, pYG1033 and pYG1033xcex94SfiI, which are described in the examples.
Compared with the prior art systems, some of the advantages of the present invention are:
the very efficient stabilisation of plasmids in yeasts of the Kluyveromyces genus;
the possibility of culturing the recombinant cells in a medium which is not very expensive and which can be easily obtained in large amounts; and
the possibility of detecting recombinant cells in a very simple manner, which renders the use of additional selection markers unnecessary. Indeed, it is generally necessary to use one or more markers in order to identify and/or select recombinant cells. Genes which confer resistance to antibiotics such as in particular geneticin (aph gene), or to other compounds which are toxic for the cell, such as copper ions (CUP gene), are most often involved. Genes complementing auxotrophies of the host cell (URA3, TRP1, or LEU2 genes and the like) may also be involved. The host/vector pairs of the invention make it possible to avoid the use of any other selection marker.
The present invention will be more completely described by means of the following examples which should be considered as illustrative and nonlimiting.